THE INABILITY OF CHOLINE TO TRANSFER A METHYL GROUP DIRECTLY TO HOMOCYSTEINE FOR METHIONINE

FORMATION

BY JOHN A. MUNTZ WITH THE TECHNICAL ASSISTANCE OF JERARD HURWITZ

(From the Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland)

(Received for publication, July 23, 1949)

Since the development of the concept of “labile” methyl groups in the metabolism of mammals, it has been apparent that choline is one of the naturally occurring sources of methyl groups. The evidence for this comes largely from two lines of research. One is the demonstration that when rats are fed choline labeled with deuterium in the methyl groups these labeled groups appear in methionine and in creatine which may be isolated from the body tissues (1). The other line of evidence comes from experiments with young rats in which it was shown that choline will sup- port growth on a methionine-free diet properly supplemented with homo- cysteine (2). Formation of methionine from choline (or betaine) and homocysteine has been stated to be one type of transmethylation (3). There remained the question of whether the choline was a direct donor of methyl groups or whether it had to be converted to another compound before methyl transfer could occur.

Further study of the transmethylation process, with liver slices and extracts, at first seemed to support the idea that choline was a direct methyl donor, since the formation of methionine from choline and homocysteine could occur anaerobically in such isolated systems (3). However, in these experiments betaine was more effective than choline for the forma- tion of methionine. The possibility that choline was converted first to betaine prior to reaction with homocysteine could not be ruled out.

More recently it has been reported that liver homogenates prepared from rabbits, guinea pigs, and chicks, which do not oxidize choline, are unable to catalyze the formation of methionine from homocysteine and choline (4). Other types of experiments which throw doubt upon the ability of choline to transfer directly its methyl groups have been done with some sulfur-containing compounds, the thetins. It has been shown that dimethylthetin and propiothetin, which are analogous to betaine and alanine betaine respectively, will cause methionine to be formed when they are incubated with homocysteine in the presence of rat liver ho- mogenates (5). They will also support the growth of young rats that are

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490 METFKION-INE FORMA!lXON

kept on a diet lacking in methionine and choline but supplemented with homocysteine (6). On the other hand, the sulfur compound analogous to choline (sulfocholine) will not support the growth of young rats kept on a similar diet (7).

HaC \ H H HHH

(a) HJ2-N’6-C-C-OH + HS-C-C-C-COO- __) /+ H H HH t

KC NH3 +

KC \ H H H H H

N’-C-C-OH + HZ-S-C-C-C-COO- / H H HH 1

HsC NH3 +

With the use of the recently described system that synthesizes methi- onine in vitro (5), it was possible to test whether choline could transfer methyl directly to homocysteine. Choline chloride was prepared which contained 30 atom per cent excess N16. It was allowed to react with homocysteine in the presence of rat liver homogenate. Since it has been shown that only one of the methyl groups of choline is labile (8), direct participation of choline in the methylation of homocysteine should yield dimethylaminoethanol which contained the N16 of the choline (a).

H& \-HH

(b) H3C-N-C-C-OH /+ H H

HZ

HaC \ H H H H

H&-N-C-COO- + HS-C-C-C-COO- - /+ H HH 1

KC NH3 +

HaC \ H H H H

H&-N’-C-COO- + H,C-S-C-C-C-COO- /+ H HH I

H NHs +

On the other hand, if choline is first oxidized to betaine before methyl transfer can occur, and if betaine is the direct donor, dimethylamino-

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J. A. MU-NT2 491

ethanol should not be formed, but instead dimethylglycine containing Nls should appear as a product of the reaction. Betaine has been shown to cause methionine formation when it is incubated together with homocys- teine and rat liver slices or homogenates (3). This is shown schematically @I.

In both of these formulations, which are written for illustrative purpose and not to indicate exact mechanisms, the reaction is written with homo- cysteine as the methyl acceptor. In the relatively crude system used, there may be other acceptors as well. Furthermore, there may be still other intermediates between choline and betaine on the one hand and the methyl acceptor on the other.

The experiments reported here show that the N’j of choline appeared in a fraction that had the characteristics of dimethylglycine, and negli- gible amounts appeared in dimethylaminoethanol.

EXPERIMENTAL

Isotopic Choline-500 mg. of NH&l containing 31 atom per cent Nl6 were made to react with 1.33 gm. of paraformaldehyde (9). The result- ing trimethylamine was distilled directly into ethylene chlorohydrin held at -30”. When all the amine had been transferred, the glass bomb tube containing the ethylene chlorohydrin was sealed and heated for 3 hours in a boiling water bath. The contents of the tube were washed out with absolute ethanol, and the choline chloride was precipitated by adding 10 volumes of absolute ether. It was purified by repeated solution in absolute ethanol and reprecipitation with ether. A small sample (70 mg.) was prepared for analysis in the mass spectrometer (10) and was found to contain 30.12 atom per cent excess Nr6.

Synthesis of Methionine in Liver Homogenates-Homogenates were made from the livers of rats fasted 24 hours by homogenizing 1 part of liver with 4 parts of phosphate buffer in an all-glass homogenizer. The buffer had the following composition: 0.0128 M sodium phosphate, pH 7.4; 0.123 M NaCl; 0.005 M KCl; 0.0033 M MgS04; and 0.02 M NaHC03. Five large Warburg type vessels were set up as follows: 3.75 ml. of homogenate, 11.25 ml. of buffer, 2.5 ml. of buffer containing 3.5 mg. of homocysteine hydrochloride, and 2.5 ml. of solution containing 5.6 mg. of choline chlo- ride in a side arm. The vessels were gassed throughout the 4 hour incu- bation period with Nz or 95 per cent Nz-5 per cent CO2 or combinations of these gases. They were incubated at 37” with continuous gentle shak- ing. The solution of choline chloride was tipped into the main compart- ment after an initial 4 minute gassing period. After incubation, the con- tents of the flasks were pooled, and each flask was washed out with 5 ml. of HZ0 which was added to the total volume. Trichloroacetic acid (12.5

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492 METHIONINE FORMATION

ml. of a 45 per cent solution) was then added to deproteinize the mix- ture. Methionine determinations were made on 2 ml. samples of filtrate by a sensitive modification of the McCarthy-Sullivan method (5). To the remaining filtrate, 125 mg. of sodium dimethylglycine (DMG) and 0.2 ml. of dimethylaminoethanol (DAE) were added as carriers for the small amounts of these substances which might have been formed during the incubation period. In some experiments neutralized DAE was added with the choline prior to incubation; thus any small amount of DAE formation from choline would immediately mix with the large pool of added DAE, and the formed DAE would be present in the residual DAE even though there was a secondary conversion of part, of it t’o other prod- ucts.

Separation of DAE from DMG in Filtrate-The trichloroacetic acid filtrate was concentrated to about 10 ml. in vacua and the concentrate was made 0.2 N with respect to HCl. It was then extracted eight times with 10 ml. portions of ether to remove trichloroacetic acid. The ex- tracted solution was adjusted to pH 9 and then was made 0.1 N with re- spect to NaOH. This alkaline solution was extracted with ether in a continuous extractor for G hours. The ether phase contained 2.5 ml. of 1 N HCl to convert the amine into its hydrochloride. The extracted alkaline solution was saved for DMG isolation.

Isolation of DAE from Ether Phase-Ether was evaporated from the solution on a steam bath, and the amine hydrochloride was concenkated to approximately 0.5 ml. It was then treated with a small amount of anhydrous CaO; thus the resulting mixture appeared dry and crumbly. This mixture was then extracted continuously for 4 hours with ether into 200 ml. of ether’ containing 0.55 gm. of picrolonic acid (11). Dimethyl- aminoethanol picrolonate began to separate out immediately, and nearly quantitative yields were obtained. The precipitate was collected on a filter and was recrystallized five or six times from absolute ethanol. Its melting point was found to be 196.5-197.3” (corrected). The melting point has been shown to be about 197” (12).

Separation and Decomposition of DMG Fraction-While it was possible to separate DAE from the mixture as the picrolonate, which is a well characterized derivative, no success was achieved in preparing a good derivative of DMG. Consequently, the following rather long purification procedure was devised. The extracted alkaline solution was centrifuged, and the precipitate was discarded. The supernatant was adjusted to pH 6.5 with 5 N HCl and the mixture was dried in vacua. The resulting dry salt was extracted five times with 25 ml. portions of boiling absolute

1 Squibb’s U. S. P. ether for anesthesia was used to dissolve the picrolonic acid; absolut,e ether proved to be unsatisfactory for this purpose.

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J. A. MUNTZ 493

alcohol, and the combined extracts were filtered and again dried in vacua. The residue, dissolved in 10 ml. water, was adjusted to pH 7 and was then passed through a 6 X 100 mm. column of Amberlite IRC-50 resin, sodium form2 The eflluent was treated wit.h 5 ml. of 30 per cent NaNOn and 3 ml. of glacial acetic acid at room temperature for 10 minutes, and the mixture was then heated to 90” for 30 minutes with continuous aera- tion to decompose the nitrous acid and to remove part of the acetic acid. Following this procedure, the flask was cooled, and the mixture was re- adjusted to pH 7 with 10 N NaOH. 3 ml. of a 20 per cent Mg(OH)2 suspension were added and the mixture was aerated again for 50 minutes. The entire mixture was treated with 6.6 mM of Ag,O in the manner described by Herbst and Clarke (13). Dimethylamine derived from di- methylglycine was caught in 5 ml. of ice-cold 0.2 N H&Sod. It was nec- essary to remove from the DMG fraction as many as possible of the nitrogenous products which might contain N15. These would include be- taine and betaine aldehyde (14), as well as unchanged choline, since it has been shown that rat liver preparations catalyze the oxidation of choline t.o these products. Choline and betaine aldehyde are removed quanti- tatively by the slightly alkaline IRC-50 column. It is necessary to work with an alkaline column since HCl is liberated when choline chloride is adsorbed on the resin as furnished by supply houses. If this were to oc- cur, dimethylglycine would also be retained on the column. From the alkaline column, DMG and betaine pass quantitatively into the effluent. Attempts to use phosphotungstic acid and Reinecke salt for separating these two substances were unsuccessful, despite the fact that betaine is readily precipitated with Reinecke salt, while DMG is not. Apparently a coprecipitation takes place.

However, DMG and betaine behave quite differently when they are treated with a silver oxide suspension at 1OOo.3 When DMG is boiled gently in a mixture of Ag,O and Mg(OH)z, a nearly quantitative yield of dimethylamine is obtained, and this may be removed by aeration of the mixture with either hydrogen or nitrogen. On the other hand, betaine is completely stable under these conditions (13). Under the same circum- stances, primary amino acids react to liberate NH,; consequently, nitrous acid was used to destroy these amino acids. It was found that when the nitrous acid-treated mixture was neutralized and Mg(OH), was added some NH3 was liberated. Therefore, the mixture was aerated for 50 min-

2 A suspension of Amberlite IRC-50, as furnished by the Fisher Scientific Company, was t,reated with 5 N YaOH while stirring continuously. When the pH of the mix- ture remained constant at S.0, the mixture was filtered, and the resin was washed with distilled water and stored damp.

3 The author thanks Dr. David U. Sprinson for suggesting this method of sepsrat- ing betaine from dimethylglycine.

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494 METHIONINE FORMATION

utes prior to AgzO treatment. This final aeration was not carried out in Experiments 1 to 3 of Table I.

TABLE I

N16 Content of Various Nitrogenous Fractions Isolated from Reaction Mixture*

Amount of carrier addedt

DAE DMG

Total methionine synthesized

ml. w. Y

0.2 100 146 0.2 100 321 0.2 125 962 0.2 125 642 0.2 100 560 0.1 125 706 0.1 125 1025 0.1 125 800

-

Atom per cent excess N’s in

Dimethyl- rminoethanol

- I

r

+action con- taining di-

nethylglycine

0.009 0.094 0.05 0.288 0.008 0.269 0.004 0.336 0.009 0.32 0.032 0.417 0.00 0.224 0.00 0.202

Volatile nitrogenous

fraction -~

0.159 0.167

In Experiment 1 the homogenate-buffer mixture was gassed for 5 minutes with 95 per cent Nz-5 per cent COZ, then incubated for 3.5 hours at 38”. Carriers DAE and DMG were added after the incubation period. In Experiment % the homogenate- buffer mixture was gassed continuously with 95 per cent Ns-5 per cent COn through- out a 4 hour incubation period. Carriers DAE and DMG were added after the in- cubation period. For Experiment 9 the homogenate-buffer mixture was gassed with Nz for 5 minutes, and the side arm tap was then left open to the air for 1 hour. The vessels were gassed with Ne for 2 hours and finally with pure CO2 for 1 hour. Car- riers DAE and DMG were added after the incubation period. For Experiment ,$ the same gassing procedure was employed as in Experiment 3. Carrier DAE was added at the start of the experiment; carrier DMG was added after the incubation period. In Experiment 6 the homogenate-buffer mixture was gassed with N1 for 5 minutes; the taps were closed and the mixture was incubated for 3.5 hours. Car- riers DAE and DMG were added after the incubation period. For Experiment 6 the homogenate-buffer mixture was gassed with N) continuously for 1 hour, then with 95 per cent Nz-5 per cent CO, for 3 hours. Carrier DAE was added at the start; DMG was added after the incubation period. In Experiment 7 the homog- enate-buffer mixture was gassed with N) continuously for 1 hour, then with 95 per cent Nz-5 per cent CO2 for 3 hours. Carrier DAE was added at the start; DMG was added after the incubation period. The same conditions were employed in Experiment 8 as in Experiment 7.

* 100 ml. of homogenate-buffer containing 17.5 mg. of homocysteine hydrochlo- ride and 28 mg. of choline chloride.

t DAE, Eastman Kodak &dimethylaminoethyl alcohol. DMG, sodium di- methyl-glycine.

Preparation of Nitrogen Samples for Mass Spectrometer-The DAE pic- rolonate was decomposed with 7 ml. of N HzS04 in a boiling water bath and after cooling, was extracted by three shakings with 2 volumes of

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J. A. IKUNTZ 495

ethyl acetate to remove picrolonic acid (11). The clear, colorless amine solution was then digested with an HzS04-CuSOd-selenium Kjeldahl di- gestion mixture for 16 hours. Dimethylamine sulfate solution derived from the dimethylglycine fraction was dried in an oven at 100” and like- wise was digested for 16 hours. The Kjeldahl digests were steam-distilled into 0.5 ml. of 4 N H$304; the nitrogen was liberated with hypobromite reagent and was transferred to mass spectrometer sampling tubes (10).

Results

As one of the controls on the methods employed in this study, a filtrate was prepared from an incubated homogenate mixture to which no choline had been added. No DAE or DMG was added, and the entire procedure was carried out to see whether any volatile basic substances were formed during the final AgzO oxidation step. A small amount of such substances was formed, equivalent to 0.487 mg. of N in one experiment. This nitro- gen would dilute the isotopic nitrogen derived from DMG in our experi- ments. It was confirmed that betaine did not react with AgzO either in pure solution or in the presence of extract prepared from liver filtrate, nor did it break down in any of the other chemical manipulations.

As shown in Table I, DAE isolated from the liver homogenate filtrate usually contained no significant amount of N15. This was so regardless of whether carrier DAE was added at the beginning or at the end of the incubation period. Carrier DAE was added at the start of the experiment in order to have isotopic DAE, if it were formed from choline, mix imme- diately with the large non-isotopic pool. In this way even if part of the DAE were oxidized to DMG, the residual DAE isolated at the end of the experiment should contain some isotope. Actually the DAE added was nearly quantitatively recovered in all cases. In Experiments 2 and 6, a small amount of N15 was detected. The amount of N15 found was small by comparison with the amount of isotope found in the DMG fraction in the same experiment. To check the possibility that isotopic choline might break down to yield a small amount of amine in the course of the chemical manipulations, the following experiment was carried out. A liver ho- mogenate was incubated with buffer in the usual way. Immediately after adding trichloroacetic acid, 25 mg. of isotopic choline chloride were added, together with 0.15 ml. of DAE and 125 mg. of DMG. These sub- stances were then isolated from the filtrate in the manner described pre- viously. Neither the DAE nor the DMG fraction contained a significant amount of N15 (Experiment 12, Table II). This shows that choline is stable under t.he conditions of the ether extraction and is removed quanti- tatively by IRC-50 resin.

Such small amounts of isotope as appeared in the DAE in some experi-

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496 METHIONINE FORMATION

ments could be due to NH, or amines which might be formed as breakdown products of DMG. There are enzymes present in liver which convert DMG to sarcosine and this in turn may be converted to glycine (15). Sarcosine also may be enzymatically degraded to methylamine by glycine oxidase, and the same enzyme converts glycine to NH, and glyoxylic acid (16). Although these are aerobic processes, other hydrogen acceptors will promote these oxidations. Methylamine and NH, would form picrol- onates which might be difficult to separate from the picrolonate of DAE

TABLE II Control Experiments on Stability of Choline Containing N’s, with and without

Experiment No.

10 11 12 13

Incubation

Amount of carrier added / Atom per cent excess N’s in

-7

DAE

ml.

0.1 0.1 0.15 0.2

DMG DAE DMG

%.

125 0.00 125 0.00 125 0.00

11*

0.03 0.044 0.008 0.016

Volatile nitrogenous

fraction

O.OG6 0.150

In Experiment 10 the homogenate-buffer-choline mixture wa8 incubated with- out homocysteine, gassed with Nf for 1 hour, then with 95 per cent X2-5 per cent COn for 3 hours. Carrier DAE was added at the start of the experiment, while DMG was added to the filtrate. The conditions of Experiment 11 are the same as in Experiment 10. In Experiment 1.2 the homogenate-buffer mixture was incubated for 4 hours without homocysteine or choline. N*6-Choline chloride, DMG, and DAE were added to the filtrate, and the fractions were isolated in the usual manner. For Experiment 13 the same general procedure was used as in Experiment 12, except that Nl6-choline chloride was added after the DAE had been extracted and no carrier DMG was added.

* Sodium dimethylglycine added to provide enough nitrogen for mass spectrom- eter analysis.

by recrystallization. If CHZNHZ andNH3 were formed, it should be pos- sible to remove them from the alkaline solution prior to ether extraction. In some of the later experiments this was done by aerating the alkaline solution vigorously for 20 minutes. The volatile substances were caught in 0.1 s H2’SOa, converted to (NH&SO4 by Kjeldahl digestion, and then analyzed for N15. As is shown in Experiments 7 and 8, Table I, this frac- tion contained a significant amount of isotope, and the DAE which was removed subsequently contained no isotope.

In contrast to the negligible amounts of isotope usually found in the DAE, the DMG fraction always contained large amounts of N16. The

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J. A. MUNTZ 497

quantity found was usually greater than the amount that would be ex- pected from the methionine synthesized. Thus, in Experiment 2, 0.081 atom per cent excess N*5 should have appeared in the DMG fraction if DMG equivalent to 321 y of methionine had been formed. The extra amount of N16 which was found might be accounted for in at least two ways: (a) the methionine found by analysis does not represent a11 the methionine that was formed; (b) betaine contributed a methyl group to acceptors other than homocysteine, thus yielding an augmented amount of DMG.

Some evidence for a slight destruction of methionine was obtained, but the analytical method for methionine determination is not sufficiently sensitive or precise to make this certain. Liver slices will metabolize methionine by deaminizing it to the corresponding keto acid (17). In- direct evidence that betaine can yield some DMG in the absence of added homocysteine was obtained by carrying out a 4 hour experiment with the usual amount of N15-labeled choline but with no homocysteine present. Under these conditions a small amount of isotope appeared in the DMG fraction (Experiments 10 and 11, Table 11). A similar experiment, in which the choline was added after the addition of trichloroacetic acid to the homogenate, showed that choline N15 did not appear in the DMG fraction (Experiment 12, Table 11).

A11 of the results make it quite certain that the formation of the DMG is in the main related to a reaction with homocysteine, and that the DMG is not a product of a reaction unrelated to methylation of homocysteine.

DISCUSSION

In none of these experiments was there an appreciable amount of N15- labeled DAE formed when N16-labeled choline chloride and homocysteine were incubated together with rat, liver homogenate. Consequently, re- action (a) did not take place in this system. On the other hand, the relatively high isotope content in the fraction derived from DMG suggests that reaction (b) represents the manner in which methyl transfer from choline takes place; however, there may still be other intermediates in the transfer of the methyl group from betaine to homocysteine. The finding that DMG is the product of the reaction is in agreement with experiments in vivo in which betaine was shown to yield methyl groups for the synthesis of choline and creatine (18). In this same paper it was shown that betaine is not converted as a whole to choline; consequently, the reaction choline -+ betaine is irreversible, a fact which had previously been demonstrated with N15-labeled betaine (19).

Since most of these experiments were carried out under anaerobic con- ditions, it is necessary to assume that choline oxidation is facilitated by hydrogen acceptors present in the liver homogenate. An anaerobic con-

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498 METHIONINE FORMATION

dition was maintained so as to give every opportunity for methyl transfer from choline to occur and to retard the oxidation of choline to betaine. Many small scale experiments were performed to find the conditions udder which uniformly maximal amounts of methionine were formed. Early experiments were carried out in phosphate buffer at pH 7.4 and the results were erratic. When bicarbonate was incorporated into the buffer and the vessels were gassed initially with Nz, thus giving pH 7.8, it was found that methionine formation was greater and more uniform. Presumably the higher pH favors formation of betaine, whereas at the lower pH betaine aldehyde is produced (20). During the last 2 or 3 hours of the incubation period, the gas mixture was changed to 95 per cent Nz-5 per cent COZ so as to lower the pH to 7.4. The lower pH appears to favor transfer of methyl from betaine to homocysteine.

It was also observed that better synthesis was obtained when the vessels were gassed continuously throughout the incubation period. This may be due to the removal of H&S, for it was observed that, if the vessels were not gassed continuously, a strong odor of HZS was always present.

There is not good agreement between the amount of methionine syn- thesized in these experiments and the level of N16 in the nitrogen derived from the DMG fraction. Possible reasons for this have already been presented. It is to be noted that in Experiments 10 and 11 the Nl5 level is higher in the volatile nitrogenous fraction than in the DMG fraction. This seeming paradox is explained by the fact that the isolated DMG nitrogen is diluted with 14 mg. of normal DMG nitrogen, added as carrier, while no carrier nitrogen was added to the volatile nitrogenous fraction.

This work was initiated with Dr. A. D. Welch of the Department of Pharmacology who helped with the synthesis of the Nr5-choline chloride. It is a pleasure to acknowledge his continued interest and advice.

SUMMARY

Rat liver homogenates incubated with choline and homocysteine form methionine, but dimethylaminoethanol, the expected product of the de- methylation of choline, is not produced. Instead dimethylglycine, the expected product of the demethylation of betaine, is formed. This sug- gests that in these systems choline does not lose a methyl group directly but must first be converted to betaine before methyl transfer can occur.

BIBLIOGRAPHY

1. Simmonds, S., Cohn, M., Chandler, J. P., and du Vigneaud, V., J. Biol. Chem., 149, 519 (1943).

2. du Vigneaud, V., Chandler, J. P., Moyer, A. W., and Keppel, D. M., J. Biol. Chem., 131, 57 (1939).

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3. Borsook, H., and Dubnoff, J. W., J. BioZ. Chem., 169, 247 (1947). 4. Dubnoff, J. W., Federation Proc., 8, 195 (1949). 5. Dubnoff, J. W., and Borsook, H., J. Biol. Chem., 176, 789 (1948). 6. Maw, G. A., and du Vigneaud, V., J. Biol. Chem., 176, 1037 (1948). 7. Maw, G. A., and du Vigneaud, V., J. Biol. Chem., 1’76, 1029 (1948). 8. du Vigneaud, V., Chandler, J. P., Simmonds, S., Moyer, A. W., and Cohn, M.,

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assistance of Jerard HurwitzJohn A. Muntz and With the technical

METHIONINE FORMATIONDIRECTLY TO HOMOCYSTEINE FOR

TRANSFER A METHYL GROUP THE INABILITY OF CHOLINE TO

1950, 182:489-499.J. Biol. Chem. 

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